1. Abstract Prostaglandins E1 and E2 (PGE1 and PGE2) bind to four receptors, designated EP1 through EP4, on the surface of mammalian cells. The EP2 and EP4 receptors are known to stimulate bone formation by activating adenylate cyclase, resulting in elevated cyclic AMP levels in osteoblasts, the cells responsible for forming bone. Binding of PGE2 to EP4 is also known to cause a population of bone marrow cells to become adherent and differentiate into osteoblasts. Ultimately, this project will further investigate the differentiation of bone marrow cells into osteoblasts at the molecular level. Our first goal was to clone and express the rat EP2 and EP4 receptors in cells to establish a reporter assay for the elevation of cyclic AMP following the binding of the ligand to the receptor. Rat EP2 and EP4 had been cloned from rat kidney cDNA into the pCMV Script expression plasmid. Conditions are being determined for transfection of the DNA into Cos-7 cells using CMV ｧ - galactosidase, since this enzyme can be detected using a colormetric assay. Once optimal conditions are determined, we will transfect each receptor DNA together with a cyclic AMP responsive reporter plasmid to demonstrate binding of ligand and receptor activation. Once established using PGE2 as a positive control, other compounds such as PGA1 and PGA2 will be tested for their ability to activate these receptors. We will then begin to explore the role of these receptors in the transformation of pluripotent bone marrow cells into committed osteoblasts.
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